Direct measurement of unfractionated heparin using a biochemical assay.

A number of investigations have noted that functional biological assays for heparin are not always reliable and may not reflect the actual biochemical level of heparin in patients receiving anticoagulant therapy. This creates the possibility that patients receiving anticoagulant treatment may have an excess or deficiency of circulating levels of heparin. To address this problem, we have developed a direct biochemical measurement of heparin. The heparin assay uses fluorophore-assisted carbohydrate electrophoresis (FACE) to directly measure the predominate disaccharide of unfractionated heparin. In this study, unfractionated heparin was measured in vitro throughout a wide range of heparin concentrations in plasma. Seven in vivo pharmacokinetic studies in five normal subjects given 3,000 USP units of unfractionated heparin intravenously showed a three-phase elimination process with higher peak plasma levels and shorter elimination times than predicted from previous studies. At these doses, heparin is largely eliminated intact through urinary excretion. Body weight has a significant effect on heparin kinetics. When we compared the direct biochemical assay with two biological clotting assays, we found the latter can overestimate biochemical heparin concentrations. The FACE assay, due to its sensitivity, is also able to measure circulating levels of endogenous heparin in plasma and urine. Direct heparin measurement using the FACE technique is practical and useful for studies of the correlation of biochemical and biological activities.

Human mononuclear phagocyte inducible nitric oxide synthase (iNOS): analysis of iNOS mRNA, iNOS protein, biopterin, and nitric oxide production by blood monocytes and peritoneal macrophages.

Nitric oxide (NO) is produced by numerous different cell types, and it is an important regulator and mediator of many processes including smooth muscle relaxation, neurotransmission, and murine macrophage-mediated cytotoxicity for microbes and tumor cells. Although murine macrophages produce NO readily after activation, human monocytes and tissue macrophages have been reported to produce only low levels of NO in vitro. The purpose of this study was to determine if stimulated human mononuclear phagocytes produce inducible nitric oxide synthase (iNOS) mRNA, protein, and enzymatic activity. By reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, we show that human monocytes can be induced to express iNOS mRNA after treatment with lipopolysaccharide (LPS) and/or interferon-gamma (IFN-gamma). By immunofluorescence and immunoblot analyses, we show monocytes and peritoneal macrophages contain detectable levels of iNOS antigen after stimulations with cytokines in vitro. Control monocytes or those cultured with LPS and/or various cytokines have low levels of NOS functional activity as measured by the ability of cell extracts to convert L-arginine to L-citrulline, and they produce low levels of the NO catabolites nitrite and nitrate. Peritoneal macrophages have significantly enhanced nitrite/nitrate production and NOS activity after treatment with LPS and/or IFN-gamma, whereas monocyte nitrite/nitrate production and NOS activity are not altered by the treatments. Monocytes cultured with various live or heat-killed bacteria, fungi, or human immunodeficiency virus (HIV)-1 do not produce high levels of nitrite/nitrate. Antibodies against transforming growth factor-beta (TGF-beta), a factor known to inhibit iNOS expression and NO production in mouse macrophages, do not enhance NO production in human monocytes or macrophages. Biopterin, an obligate cofactor of iNOS enzymatic activity, is undetectable in freshly isolated or cultured human monocytes and peritoneal macrophages. However, replenishment of intracellular levels of tetrahydrobiopterin by culture with the cell-permeable, nontoxic precursor sepiapterin does not enhance the abilities of the human mononuclear phagocytes to produce NO in vitro. Mixing experiments show no evidence of a functional NOS inhibitor in human mononuclear phagocytes. Thus, we demonstrate that human mononuclear phagocytes can produce iNOS mRNA and protein, and (despite this) their abilities to generate NO are very low.

Characterization of thrombomodulin expression in response to retinoic acid and identification of a retinoic acid response element in the human thrombomodulin gene.

Thrombomodulin (TM) is an essential cofactor for the physiologic activation of the anticoagulant protein C by thrombin. We have observed that the expression of TM mRNA in response to retinoic acid was markedly increased in human U937 monoblast-like cells, and human MEG01 megakaryocyte-like cells, but not in human umbilical vein cells, murine hemangioma cells, human K562 erythroblast-like cells, and murine HSD fibroblast-like cells. TM activity in U937 cells and MEG01 cells was not detectable in untreated cells, but developed rapidly after treatment with retinoic acid. In endothelial cells there was minimal change in TM activity in response to retinoic acid treatment. We have isolated clones for the genes for murine and human TM and have identified potential retinoic acid response elements in the 5'-flanking region of the human gene. In U937 cells the increase in mRNA levels was associated with increased transcription, and transient transfection studies with reporter plasmids demonstrate functional retinoic acid response elements present in the 5'-flanking region of the gene. Deletion of, and mutations introduced into, the potential retinoic acid response element confirm the functional response in transient transfections.

Thrombomodulin expression by human blood monocytes and by human synovial tissue lining macrophages.

Thrombomodulin is an essential cofactor for the activation of the anticoagulant protein C by thrombin. We have identified the expression of thrombomodulin messenger RNA (mRNA) and protein in peripheral blood monocytes. While untreated monocytes expressed thrombomodulin mRNA by Northern blot analysis, lipopolysaccharide-treated cells had decreased mRNA expression. Thrombomodulin antigen was shown in the cytoplasm and on the surface of monocytes by immunohistochemical staining, and thrombomodulin activity was shown on the surface of intact monocytes. One population of synovial lining cells that normally expressed mononuclear phagocyte antigens also expressed thrombomodulin in both noninflamed osteoarthritic synovium and in inflamed rheumatoid arthritis synovium. However, these cells did not express another endothelial protein, von Willebrand factor. We conclude that both circulating and tissue mononuclear phagocytes are capable of expressing thrombomodulin.

Increased expression of thrombomodulin on the cultured human umbilical vein endothelial cells and mouse hemangioma cells by cyclic AMP.

We previously reported that the expression of thrombomodulin on the MEG-01, a cell line from human megakaryoblastic leukemia, was increased by agents that increase intracellular cAMP. In this paper we examine the effect of these agents on cultured human umbilical vein endothelial cells (HUVEC) and mouse hemangioma cells. Incubation of the cells with 3 mM dibutyryl cAMP (dbcAMP) increased functionally active thrombomodulin by about 2-fold on HUVEC and 4-fold on hemangioma cells. This effect was observed from 1 hour after the incubation and continued up to 24 hours. Dot hybridization of mRNA demonstrated a dose dependent increase in thrombomodulin mRNA in response to dbcAMP. Treatment of HUVEC with 20 microM forskolin or 100 microM isobutylmethylxanthine (IBMX) also increased cell-surface thrombomodulin on HUVEC. These agents prevented the interleukin I (IL-I) or tumor necrosis factor (TNF)-induced decrease in thrombomodulin on HUVEC. These data suggest that the expression of thrombomodulin on HUVEC and mouse hemangioma cells may be regulated by intracellular cAMP level.

Thrombomodulin Biology and potential cardiovascular applications.

Thrombomodulin on endothelial surfaces is the major cofactor for the activation of the anticoagulant protein C by thrombin. Recent studies of the structure and regulation of thrombomodulin provide important clues as to its mechanism of action. These studies should enable the rational use of thrombomodulin as a therapeutic agent to inhibit coagulation either by infusion of thrombomodulin or its derivatives, or by pharmacologic alteration of cellular levels.

Equilibrium binding of thrombin to recombinant human thrombomodulin: effect of hirudin, fibrinogen, factor Va, and peptide analogues.

Thrombomodulin is an endothelial cell surface receptor for thrombin that acts as a physiological anticoagulant. The properties of recombinant human thrombomodulin were studied in COS-7, CHO, CV-1, and K562 cell lines. Thrombomodulin was expressed on the cell surface as shown by the acquisition of thrombin-dependent protein C activation. Like native thrombomodulin, recombinant thrombomodulin contained N-linked oligosaccharides, had Mr approximately 100,000, and was inhibited or immunoprecipitated by anti-thrombomodulin antibodies. Binding studies demonstrated that nonrecombinant thrombomodulin expressed by A549 carcinoma cells and recombinant thrombomodulin expressed by CV-1 and K562 cells had similar Kd's for thrombin of 1.3 nM, 3.3 nM, and 4.7 nM, respectively. The Kd for DIP-thrombin binding to recombinant thrombomodulin on CV-1(18A) cells was identical with that of thrombin. Increasing concentrations of hirudin or fibrinogen progressively inhibited the binding of 125I-DIP-thrombin, while factor Va did not inhibit binding. Three synthetic peptides were tested for ability to inhibit DIP-thrombin binding. Both the hirudin peptide Hir53-64 and the thrombomodulin fifth-EGF-domain peptide Tm426-444 displaced DIP-thrombin from thrombomodulin, but the factor V peptide FacV30-43 which is similar in composition and charge to Hir53-64 showed no binding inhibition. The data exclude the significant formation of a ternary complex consisting of thrombin, thrombomodulin, and hirudin. These studies are consistent with a model in which thrombomodulin, hirudin, and fibrinogen compete for binding to DIP-thrombin at the same site.

Transcription of thrombomodulin mRNA in mouse hemangioma cells is increased by cycloheximide and thrombin.

We have measured mRNA levels for thrombomodulin, an endothelial membrane cofactor for the activation of protein C by thrombin, in a mouse hemangioma cell line. Cycloheximide, an inhibitor of protein synthesis, increased levels of thrombomodulin mRNA, as measured in an S1 nuclease protection assay, to 2.5-4.0 times control levels. Thrombomodulin transcription in response to cycloheximide treatment, as determined by nuclear run-on analysis, was 3.9 +/- 1.3 (mean +/- SD) times that found in untreated cells. Thrombin also increased thrombomodulin mRNA levels to 151 +/- 21% (mean +/- SD) of control levels after 2 hr. Transcription increased in response to thrombin by 2.1- to 7.3-fold. The combination of thrombin and cycloheximide had no additive effect on thrombomodulin mRNA levels. Thrombin treatment of hemangioma cells also caused an increase in thrombomodulin protein synthesis to 142 +/- 17% (mean +/- SD) of control levels as determined by immunoprecipitation of [32S]methionine-labeled thrombomodulin. We conclude that thrombomodulin expression is determined in part by the rate of transcription and that thrombomodulin mRNA levels in hemangioma cells are increased by treatment with cycloheximide or thrombin. The increased transcription in response to cycloheximide suggests the existence of a labile protein repressor of thrombomodulin transcription.

The structure and function of mouse thrombomodulin. Phorbol myristate acetate stimulates degradation and synthesis of thrombomodulin without affecting mRNA levels in hemangioma cells.

Thrombomodulin is an endothelial membrane anticoagulant protein that is a cofactor for protein C activation. We have evaluated the expression of thrombomodulin in cultured mouse hemangioma cells before and after treatment with phorbol myristate acetate (PMA), an agent that stimulates protein kinase C. We also isolated a cDNA encoding 481 amino acids of mouse thrombomodulin and the entire 3'-untranslated portion of its mRNA. The deduced amino acid sequence of mouse thrombomodulin is similar to those determined for human and bovine thrombomodulin. An S1 nuclease protection assay was used to measure thrombomodulin mRNA in hemangioma cells. The half-life for thrombomodulin mRNA was 8.9 +/- 1.8 h (S.D.) in cells treated with actinomycin D. Treatment with PMA had no effect on thrombomodulin mRNA levels. Thrombomodulin turnover was evaluated by immunoprecipitation of [35S]methionine-labeled thrombomodulin. The t1/2 was 19.8 +/- 3.9 h (S.D.); PMA treatment decreased the t1/2 to 10.9 +/- 1.1 h (S.D.) while increasing the rate of synthesis to a maximum of 190% of control. Protein C cofactor activity on hemangioma cells was reduced 35 +/- 4% by treatment with PMA within 30 min. This decrease was associated with a parallel decline in cell surface thrombomodulin antigen and with enhanced phosphorylation of thrombomodulin on serine residues. We conclude that thrombomodulin is phosphorylated in response to treatment of hemangioma cells with PMA which leads to decreased protein C cofactor activity and both increased degradation and synthesis of thrombomodulin.

A role for thrombomodulin in the pathogenesis of thrombin-induced thromboembolism in mice.

The antithrombotic action of thrombomodulin was studied in mice. Rat and mouse thrombomodulin were isolated from lung acetone powders, and anti-rat thrombomodulin antibodies were prepared in rabbits. The antibodies neutralized both mouse (Kd approximately 150 nM) and rat thrombomodulin (Kd approximately 50 nM). A role for thrombomodulin in vivo was shown in mice injected intravenously (IV) with thrombin. All mice injected with 15 U thrombin (bolus) died of thromboembolism (mean survival 55 minutes), whereas those injected with a lower dosage survived. Prior injection with anti-rat thrombomodulin (1.8 mg IgG/mouse) potentiated the lethal effects of subsequent thrombin, whereas injection of thrombomodulin (isolated from mouse lung) prior to thrombin prolonged survival in a thrombomodulin concentration-dependent manner. The protective effect of thrombomodulin persisted for 30 minutes but after one hour thrombin injection was as toxic as in control animals. The half life (t1/2) for plasma clearance of 125I-mouse lung thrombomodulin was nine minutes. The major site of clearance was the liver, although thrombomodulin accumulated in several organs ten minutes after injection. The mechanism by which antithrombomodulin antibodies potentiated the lethal effects of thrombin was studied by measuring the protein C activating cofactor activity on vena cava removed from animals injected with antibodies. Protein C activation was inhibited by antibodies, suggesting a role for activated protein C in prevention of lethal thromboembolism. We found no effect of antibodies on the clearance of thrombin from mouse plasma, suggesting that blockade of endothelial endocytosis of thrombin does not play a significant role in the effects of antibodies. These results indicate that thrombomodulin participates in the defense against thrombosis in vivo.

Human thrombomodulin: complete cDNA sequence and chromosome localization of the gene.

A human umbilical vein endothelial cell cDNA library in lambda gt11 was screened for expression of thrombomodulin antigens with affinity-purified rabbit polyclonal anti-thrombomodulin immunoglobulin G (IgG) and mouse monoclonal anti-human thrombomodulin IgG. Among 7 million recombinant clones screened, 12 were recognized by both antibodies. Two of these, lambda HTm10 and lambda HTm12, were shown to encode thrombomodulin by comparison of the amino acid sequence deduced from the nucleotide sequence to the amino acid sequence determined directly from tryptic peptides of thrombomodulin. Thrombomodulin mRNA was estimated to be 3.7 kilobases in length by Northern blot analysis of endothelial cell and placental poly(A)+ RNA. Thrombomodulin mRNA was not detected in human brain, HepG2 hepatoma cells, or the monocytic U937 cell line. Additional cDNA clones were selected by hybridization with the 1.2-kilobase insert of lambda HTm10. One isolate, lambda HTm15, contained a 3693 base pair cDNA insert with an apparent 5'-noncoding region of 146 base pairs, an open reading frame of 1725 base pairs, a stop codon, a 3'-noncoding region of 1779 base pairs, and a poly(A) tail of 40 base pairs. The cDNA sequence encodes a 60.3-kDa protein of 575 amino acids. The predicted protein sequence includes a signal peptide of approximately 21 amino acids, an amino-terminal ligand-binding domain of approximately 223 amino acids, an epidermal growth factor (EGF) homology region of 236 amino acids, a serine/threonine-rich segment of 34 amino acids, a membrane-spanning domain of 23 amino acids, and a cytoplasmic tail of 38 amino acids. The EGF-homology region consists of six tandemly repeated EGF-like domains.(ABSTRACT TRUNCATED AT 250 WORDS)

Electron microscopic and peroxidase cytochemical analysis of pink pseudo-Chediak-Higashi granules in acute myelogenous leukemia.

Giant round pink inclusions (congruent to 2 micrometers) were seen in neutrophilic myeloblasts, promyelocytes, and myelocytes from three patients with acute myelogenous leukemia. On preliminary examination of the bone marrow smears, these inclusions looked like ingested red blood cells in that they were pink and not azurophilic. The bone marrow specimens were processed for the electron microscopic demonstration of peroxidase with 3,3'-diaminobenzidine and H2O2 at pH 7.6. In all three cases, the inclusions were determined to be large peroxidase-positive granules since they were limited by a single unit membrane and, unlike endocytized red blood cells, were not contained within phagocytic vasuoles. The granules were homogeneously dense for peroxidase and showed no obvious crystalline structure when examined stained or unstained on grid. We believe that they correspond to the giant pink round granules Van Slyck and Rebuck observed in immature leukemic granulocytes in 1974 and termed the pseudo-Chediak-Higashi anomaly. Like the giant purple granules seen in leukemia with this anomaly, these granules also appear to be an abnormal variant of peroxidase-positive azurophil (primary) granules. Their lack of azurophilia is due to the absence of sulfated glycoaminoglycans.